Simultaneous Quantitative Determination of Amphetamines,Opiates, Ketamine,Cocaine and Metabolites in Human Hair: Application to Forensic Cases of Drug Abuse

A method using liquid chromatography–tandem mass spectrometry (LC–MS/MS) to simultaneously quantify amphetamines, opiates, ketamine, cocaine, and metabolites in human hair is described. Hair samples (50 mg) were extracted with methanol utilizing cryogenic grinding. Calibration curves for all the analytes were established in the concentration range 0.05–10 ng/mg. The recoveries were above 72%, except for AMP at the limit of quantification (LOQ), which was 48%. The accuracies were within ±20% at the LOQ (0.05 ng/mg) and between −11% and 13.3% at 0.3 and 9.5 ng/mg, respectively. The intraday and interday precisions were within 19.6% and 19.8%, respectively. A proficiency test was applied to the validated method with z-scores within ±2, demonstrating the accuracy of the method for the determination of drugs of abuse in the hair of individuals suspected of abusing drugs. The hair concentration ranges, means, and medians are summarized for abused drugs in 158 authentic cases.     

Validation of an LC–MS Method for the Detection and Quantification of BZP and TFMPP and their Hydroxylated Metabolites in Human Plasma and its Application to the Pharmacokinetic Study of TFMPP in Humans*

Abstract: An LC–MS method was developed for benzylpiperazine (BZP) and trifluoromethylphenylpiperazine (TFMPP), constituents of “party pills” or “legal herbal highs,” and their metabolites in human blood plasma. Compounds were resolved using a mixture of ammonium formate (pH 4.5, 0.01 M) and acetonitrile (flow rate of 1.0 mL/min) with a C18 column. Calibration curves were linear from 1 to 50 ng/mL (R² > 0.99); the lower limit of quantification (LLOQ) was 5 ng/mL; the accuracy was >90%; the intra‐ and interday relative standard deviations (R.S.D) were <5% and <10%, respectively. Human plasma concentrations of TFMPP were measured in blood samples taken from healthy adults (n = 6) over 24 h following a 60‐mg oral dose of TFMPP: these peaked at 24.10 ng/mL (±1.8 ng/mL) (C_max) after 90 min (T_max). Plasma concentrations of 1‐(3‐trifluoromethyl‐4‐hydroxyphenyl) piperazine peaked at 20.2 ng/mL (±4.6 ng/mL) after 90 min. TFMPP had two disposition phases (t_½ = 2.04 h (±0.19 h) and 5.95 h (±1.63 h). Apparent clearance (Cl/F) was 384 L/h (±45 L/h).     

The Pharmacokinetics of Morphine and Codeine in Human Plasma and Urine after Oral Administration of Qiangli Pipa Syrup

Papaveris pericarpium, a natural source of morphine and codeine, is the principal active component in many antitussive traditional Chinese medicines. We herein report the first PK study of papaveris pericarpium in human plasma and urine following oral administration of single (15, 30, 60 mL) and multiple dose (15 mL) of Qiangli Pipa Syrup (MOR 0.1 mg/mL, COD 0.028 mg/mL) by monitoring morphine and codeine using a HPLC‐MS/MS method. Their T_max and t_1/2 values are independent of dosages, while the AUC_0?t linearly increased with higher dosages, indicating linear PK characteristics. AUC_0?t increased obviously after multiple doses, indicating possible risk of accumulative toxicity. Urine studies suggested risks of positive opiate drug tests with a cutoff of 300 ng/mL, which lasted 6–14 h at different doses. These results provide important information for clinical safety, efficacy and rational drug use of Qiangli Pipa Syrup and also guide the related judicial expertise of its administration.     

Phenibut screening and quantification with liquid chromatography–tandem mass spectrometry and its application to driving cases

An analytical strategy for identification by an LC–MS/MS multitarget screening method and a suitable LC–MS/MS based quantification were developed for the psychotropic drug phenibut. The samples analyzed were collected during traffic control and were associated with driving under the influence of drugs. A positive sample for phenibut was identified in a single case of driving under the influence. The quantification revealed a drug concentration of 1.9 μg/mL. An interaction with blood alcohol (BAC = 0.10%) was discussed as the explanation of the way of driving and deficit manifestations observed (swaying, nystagmus, quivering of the eyelid, and reddened eyes). According to the available information, the quantified phenibut concentration could be explained by an intake of four tablets (self-reported) during the day containing 250 mg of the drug. Chromatography was performed with a Luna 5 μm C18 (2) 100 A, 150 mm × 2 mm analytical column, and a buffer system consisted of 10 mM ammonium acetate and 0.1% acetic acid (v/v) included in mobile phases marked as A (H₂O/methanol = 95/5, v/v) and B (H₂O/methanol = 3/97, v/v). An effective limit of detection (LOD = 0.002 μg/mL) could be achieved for the multitarget screening method. The quantification of phenibut was performed on a second LC–MS/MS system with LOD/LOQ values of 0.22/0.40 μg/mL. Since phenibut quantification data are rare, the presented information can be used with caution for evaluation of positive cases in the future.     

Quantitative Analysis of Gamma‐Hydroxybutyrate at Endogenous Concentrations in Hair using Liquid Chromatography Tandem Mass Spectrometry

Abstract: A method capable of quantifying endogenous concentrations of gamma‐hydroxybutyrate (GHB) in human head hair was developed and validated using liquid chromatography/mass spectrometry/mass spectrometry (LC/MS/MS). Hair was digested under alkaline conditions, and GHB was isolated using liquid–liquid extraction. LC/MS/MS was performed using atmospheric pressure chemical ionization in the negative mode, multiple reaction monitoring, and deuterated internal standard (GHB‐D₆). Linearity was observed between 0.1 and 100 ng/mg GHB (R² = 1.000). The limits of detection and quantitation in human hair were 0.2 and 0.4 ng/mg, respectively. Accuracy at 2 ng/mg and 10 ng/mg was determined to be 97% and 94%, and intra‐assay CVs at these concentrations were 5.2% and 7.4% (n = 4). Beta‐hydroxybutyrate (BHB), alpha‐hydroxybutyrate, gamma‐butyrolactone, and 1,4‐butanediol did not produce an interference, and there was negligible ion suppression or enhancement from the matrix.     

Influence of storage on larval length and age determination of the forensically important blow fly Lucilia sericata (Diptera: Calliphoridae)

One of the main tasks in forensic entomology is the determination of the minimum post-mortem interval (PMI_min) based on the age of the juvenile insects feeding and developing on the dead body. An important task is to store the evidence appropriately so that the evaluation and expert report can be used in court. However, existing recommendations can be contradictory or lacking scientific validation, e.g. by proposing various preservation liquids without knowing whether and to what extent the period of storage in such a liquid has an effect on the length of the preserved larvae. Storage time can be an issue since, due to technical and procedural circumstances, killed larvae may be stored for hours, days, weeks or even longer prior length measurement. A changed body length would have consequences for the entomological report, as the age of the larvae is usually derived from their length.This study investigates the effect of four differently concentrated ethanol solutions (70%, 80%, 90% and 96%) during a storage period of up to 196 days on the body length of stored larvae of the forensically important blow fly species L. sericata (Diptera: Calliphoridae). Larvae of different ages (24 h, 48 h and 72 h after hatching) were killed by immersion in hot, non-boiling water (≥80 °C) for at least 30 s. Their lengths were measured immediately. Subsequently samples were stored in ethanol of appropriate concentration at room temperature (approx. 22 °C). Further length measurements were made at 16 different storage intervals between 1 and 196 days.Many specimens showed a length decrease for most storage conditions and all larval ages. However, there was a tendency for 48 h- and 72 h-old larvae to increase in length after the first days of storage of up to 1.1 mm which may lead to an erroneous overestimation of the PMI_min using this kind of specimens. All changes in length within each cohort over total time were in the range of +7% to −9.1%. Significant differences in length changes within the first days of storage were found mainly in larvae stored in 70%- and 80%-ethanol, but larvae stored in 90%- and 96%-ethanol showed first significant differences on day 56 at the earliest.Our results lead to the recommendation that the measurements of fly larvae samples should be taken immediately after killing and before storage to avoid any effects. Ethanol ≥90% should be used for storage.     

Phenobarbital in hair and drug monitoring

Phenobarbital analysis was performed in vertex hair of patients by gas chromatography mass spectrometry (GC/MS). After washing with dichloromethane, about 250 mg were ground to dust in a ball mill. A 50-mg sample was stirred mechanically for 10 min with 3 ml of NH₄Cl/HCl buffer (pH 2.0) containing phenobarbital D₅. A solid phase extraction was performed (extrelut Merck) and elution was achieved with chloroform/isopropanol/n-heptane (50:17:33; v/v). A full scan (40–240 uma) acquisition was realized by GC/MS with an ion trap (ITD 700 Finnigan) using a DB5-MS chromatographic column. Quantification was achieved by integrating dominants ions (phenobarbital, 204; phenobarbital D₅, 209). Compared to serum, hair concentrates phenobarbital during anti-epileptic therapy (average value 36.4 ng/mg, n = 40 vs. 18.7 mg/l, n = 23). A group correlation exists between phenobarbital in hair and phenobarbital in serum, and between phenobarbital in hair and clinic observation in some typical cases. Phenobarbital in hair yields good information over a long period, especially when blood collection has not been made, when clinical disorders are observed on long-term therapeutic observance.     

Corporal Punishment of Children: A Multi-Generational Perspective

This paper presents a study on the self-reported usage and attitudes toward corporal punishment (CP) by a four generation sample of Jewish families in Jerusalem. The study included 655 participants: 200 adolescents, 208 young mothers, 199 old mothers, and 48 grandmothers, and tested for inter-generational and familial role differences. Results have shown that participants’ attitudes toward CP correlates significantly with age group; however, it does not correlate with family role. Implications of the results for practitioners who seek to reduce usage of CP are suggested.

Postmortem vitreous Beta-hydroxybutyrate: interpretation in a forensic setting*

Abstract: Vitreous beta‐hydroxybutyrate (BHB) was retrospectively analyzed in 1795 forensic cases using the Pointe Scientific method. Comparison of vitreous BHB with vitreous glucose in 1781 of the cases showed moderately good correlation r = 0.731. Comparison with blood alcohol levels in 1561 of the cases showed no correlation r = ?0.053. Vitreous BHB was a marker of diabetic ketoacidosis when above 6.0 mM with a vitreous glucose over 200 mg/dL. It was an indicator (>50%) for alcoholic ketoacidosis when above 6.0 mM with a vitreous glucose below 200 mg/dL. Recommendations for interpretation of vitreous BHB: <0.4 mM normal; 0.41–1.2 mM slightly elevated, rarely (<1%) of concern; 1.21–2.0 mM moderately elevated, less rarely (2.5%) of concern; 2.01–6.0 mM significantly elevated, frequently of concern (12–48%); >6.0 mM usually (100% in this study) indicated life‐threatening conditions. Vitreous BHB was helpful evaluating cases with ketogenic conditions, especially diabetes and alcoholism.     

Determination of Nicotine,Cotinine and Trans‐3′‐Hydroxycotinine using LC/MS/MS in Forensic Samples of a Nicotine Fatal Case by Oral Ingestion of e‐cigarette Liquid,

Nicotine is a potent neurotoxin alkaloid and is used in e‐cigarette liquid. The LC/MS/MS method was linear over 0.01–1.0 mg/L (r² = 0.992–0.995). Limit of detection and limit of quantitation were 0.001 mg/L (S/N = 3) and 0.003 (S/N = 10). The inaccuracy and imprecision were <13.2%. The recoveries were >99.3%. A 39‐year‐old dentist was found dead lying on the floor under the couch in his dental clinic. The concentration of nicotine, cotinine, and trans‐3′‐hydroxycotinine (heart blood/peripheral blood) was analyzed as follows: 87.2/85.2 mg/L (ratio 1.0), 1.4/1.1 mg/L (ratio 1.3), and 0.012/0.0089 mg/L (ratio 1.3), respectively. The concentration of nicotine was determined to be 6734.8 mg/kg in gastric contents and 7262.0 mg/L in remaining e‐liquid. Only, high concentration of nicotine was detected in the gastric contents as well as the two pieces of evidence collected from the death scene. This fatal case resulted from oral ingestion of e‐cigarette liquid. It is estimated that at least 714 mg of nicotine was orally ingested.     

Incorporation of propyphenazone in beard hair of a migraine patient

The incorporation of propyphenazone in beard hair after consumption of this substance present in the analgesic Migraine-Kranit (Codali) was investigated. Because of a migraine attack a volunteer took four tablets of Migraine-Kranit (one tablet contains 150 mg propyphenazone) the first day and two tablets the second day. Shaved beard hair was collected 48, 72, 96 and 120 h after the first consumption of the analgesic drug. These hair specimens were washed (acetone and water), pulverized and then incubated during 2 h in a thioglycolic solution. After solid-phase extraction on C18 columns, propyphenazone was assayed in these extracts by GC/MS operating in selected ion monitoring mode (m/z 230, 215). Diazepam-d5 was used as an internal standard. In hair specimen 1 (48 h after consumption) the highest concentration was found (170 pg/mg hair). In hair specimen 2 (72 h) and 3 (96 h) the concentration were significantly lower (44 and 18 pg/mg, respectively). After 120 h no propyphenazone could be detected (limit of detection: 5 pg/mg hair). These results show that propyphenazone was already in beard hear 2 days after consumption, whereas no more presence could be shown after 120 h. As the time period of 2 days is too short to allow entrapment into the hair matrix from bloodstream and growing of hair out of the follicle, our results suggest that incorporation of propyphenazone may be mainly due to excretion in sweat and subsequent incorporation into the hair.     

Fentanyl transdermal patches: Extraction and evaluation of a novel disposal method using NarcX®

Currently, there is no known commercially available product for disposing of used fentanyl transdermal patches. To eliminate the potential for harm and abuse, a proper disposal method is needed–one that neutralizes the dangerous amount of residual fentanyl that remains after therapeutic use of the fentanyl patch. The patent-pending liquid solution of activated carbon, known as NarcX^®, was investigated as a potential fentanyl adsorbing agent. In order to determine the amount of fentanyl remaining after a patch is treated with NarcX^®, here, we utilized hexanes to first dissolve the patch adhesive and then followed with liquid-liquid extraction with methanol to recover the fentanyl. Using liquid chromatography coupled to tandem mass spectrometry (LC/MS-MS), the extracts obtained with this method yielded between 85% and 117% recovery of fentanyl from new and unused patches. Further optimization of this method allowed for a quantitative evaluation of NarcX^®-treated fentanyl patches. 100 µg/h Apotex brand fentanyl patches were exposed to NarcX^® for 1, 24, 48, and 72 h. NarcX^® was shown to adsorb fentanyl from the patches with varying degrees of success, demonstrating an average of 66.98 ± 0.75% fentanyl adsorption after 72 h. These findings suggest that more work is needed to successfully neutralize the fentanyl patches in their entirety using NarcX^®; however, this work does demonstrate proof of concept.     

Comparing the Recognition Memory Test and the Word Choice Test in a Mixed Clinical Sample: Are They Equivalent?

The Recognition Memory Test (RMT) was compared to the Word Choice Test (WCT) within the same fixed neuropsychological battery administered to a mixed clinical sample of 237 adults to empirically evaluate the psychometric equivalence of these two instruments. On average, there was a 3-point difference in raw scores between the two instruments (M _RMT?=?44.3, SD _RMT?=?6.8; M _WCT?=?47.1, SD _WCT?=?4.6; p?d?=?.48). The probability density functions differ substantially at the two ends of the scale but are similarly ≤42. Cross-validation analyses suggest that the RMT cutoff of ≤42 is functionally equivalent to a WCT score of ≤45.     

Estimating postmortem interval for human cadavers in a sub‐tropical climate using UV‐Vis‐near‐infrared Spectroscopy

Estimating postmortem interval (PMI) of surface found skeletal remains is challenging. This novel study used UV‐Vis‐NIR spectroscopy to scan soil collected from cadaver decomposition islands (CDIs) ranging from 15‐ to 963‐d postmortem and control soils. A decomposition product spectra model (DPS model) was constructed by deducting the control soil spectra from the CDI soil spectra for the estimation of postmortem indices: PMI (d), ADD₄, ADD₁₀, and ADD₂₀. The DPS model (n = 55) was calibrated and subjected to a full cross‐validation. Calibration R² and RPD for the DPS model ranged from 0.97 to 0.99 and from 6.1 to 9.9, respectively, for the four postmortem interval indices. Validation R² and RPD for the DPS model ranged from 0.73 to 0.80 and from 1.9 to 2.2, respectively. The DPS model estimated postmortem intervals for three test CDIs in a clay soil under perennial grassland (test set 1; n = 3) and six CDIs in a sandy soil under a loblolly pine forest (test set 2; n = 6). Test set 1 had PMI prediction ranges from ?69 to ?117 days, ?796 to +832 ADD₄, +552 to +2672 ADD₁₀, and ?478 to ?20 ADD₂₀ of observed PMI. Test set 2 PMI prediction ranged from ?198 to ?65 days, ?9923 to +2629 ADD₄, ?6724 to +1321 ADD₁₀, and ?2850 to +540 ADD₂₀ of observed PMI. Test set 2 had poor predictions for two CDIs, for all measures of postmortem indices resulting in discussion of sampling depth, effect of body mass index (BMI), and scavenging.     

腹腔连续注射低剂量氯胺酮后大鼠海马LC3和Beclin1的表达

目的研究腹腔连续注射低剂量氯胺酮后大鼠海马自噬相关蛋白LC3、Beclin1的表达及其意义。方法 SD大鼠30只随机分为用药组和对照组,用药组大鼠以5mg/kg的剂量腹腔注射氯胺酮,每间隔30min 1次,共5次。对照组予以等量生理盐水。用药组大鼠按用药后时间不同分为6小组,分别于末次给药后1,3,6,12,24,48h后取海马组织备用,用免疫荧光技术和Western blot技术检测大鼠海马组织中LC3、Beclin1的表达,应用统计学处理,比较用药组与对照组的蛋白表达差异。结果与对照组比较,用药组大鼠海马组织中LC3Ⅱ/LC3Ⅰ值在1h表达开始增多,6h呈强表达,Beclinl的表达在6h开始增多,12,24,48h都呈强表达(P<0.05)。结论腹腔连续注射低剂量氯胺酮能促进海马组织发生自噬,自噬增强是对氯胺酮毒性的反应。     

Determination of manganese in human brain samples

A method is presented for the determination of manganese (Mn) in human tissue samples (especially brain) by graphite furnace atomic absorption spectrophotometry (GFAAS). After complete digestion by a mixture of concentrated nitric acid (HNO₃)/concentrated perchloric acid (HClO₄) (50:50, v/v), the samples are assayed on a Perkin-Elmer 5100 PC apparatus, equipped with transversal graphite tubes and a Mn-specific hollow cathode lamp. The furnace conditions are as follows (for each step: temperature (°C)/ramp (s)/duration (s)) dry 120/1/40; char 1200/5/10; atomization 2250/0/4; pyrolysis 2400/1/1. Zeeman correction is employed. The method is linear over the range 0.05 to 5.00 μg/g wet tissue, and the limit of detection for Mn is about 0.01 μg/g wet tissue. This simple and rapid method may be of value for the post-mortem assessment of Mn accumulation in brain structures due to occupational or iatrogenic exposure. An application is presented in which elevated levels of Mn were determined in the brain samples of a 63-year-old female deceased after long-term total parenteral nutrition involving Mn supplementation.     

The polymorphism of transferrin by ultrathin-layer isoelectric focusing. Tf phenotypes and TfC subtypes in the population of Padua

The distribution of Tf phenotypes in the population of Padua was investigated by ultrathin-layer isoelectric focusing. In our sample (n = 618) nine phenotypes, Tf C₁, C₂, C₃, C_3?1, C_2?1, C_3?2, C₁B, C₂B and C₁D, were observed and the following frequencies calculated: TfC₁ = 0.77837; TfC₂ = 0.1804; TfC₃ = 0.03641; TfB = 0.0040; TfD = 0.0008. These gene frequencies have been compared to those found in other populations. Analysis of 101 mother-child pairs was in agreement with an autosomal codominant mode of inheritance.     

Lipophilicity of fentalogs: Comparison of experimental and computationally derived data

Although fentanyl and a small number of derivatives used for medical or veterinary procedures are well characterized, physiochemical properties have not been determined for many of the newer fentanyl analogs. Partition coefficients (Log P) were determined for 19 fentalogs using the shake-flask method and liquid chromatography–tandem mass spectrometry (LC–MS/MS). Experimentally determined partition coefficients were compared with computationally derived data using six independent software sources (ACD/LogP, LogKOWWIN v 1.69, miLogP 2.2, OsirisP, XLOGP 3.0, ALogPS 2.1). Fentalogs with a wide variety of structural modifications were intentionally selected, yielding Log P values ranging from 1.21 to 4.90. Comparison of experimental and computationally derived Log P values were highly correlated (R² 0.854–0.967). Overall, substructure-based modeling using fragmental methods or property-based topological approaches aligned more closely with experimentally determined Log P values. LC–MS/MS was also used to estimate pK_a values for fentalogs with no previously reported data. Lipophilicity and pK_a are important considerations for analytical detection and toxicological interpretation. In silico methods allow the determination of physicochemical information prior to certified reference materials being readily available for in vitro or in vivo studies. Computationally derived data can provide insight regarding physiochemical characteristics of future fentalogs and other classes of synthetic analogs that have yet to emerge.     

Developmental Validation of the Quantifiler® Duo DNA Quantification Kit for Simultaneous Quantification of Total Human and Human Male DNA and Detection of PCR Inhibitors in Biological Samples*

Abstract: The Quantifiler^® Duo DNA Quantification kit enables simultaneous quantification of human DNA and human male DNA as well as detection of inhibitors of PCR in a single real-time PCR well. Pooled human male genomic DNA is used to generate standard curves for both human (ribonuclease P RNA component H1) and human male (sex determining region Y) specific targets. A shift in the cycle threshold (C_T) values for the internal positive control monitors the presence of PCR inhibitors in a sample. The assay is human specific and exhibits a high dynamic range from 0.023 to 50 ng/μL. In addition, the multiplex assay can detect as little as 25 pg/μL of human male DNA in the presence of a 1000-fold excess of human female DNA. The multiplex assay provides assessment of the DNA extract and guidance for the selection of the appropriate AmpFℓSTR^® Amplification Kit to obtain interpretable short tandem repeat profiles.     

Distribution of Chemical Phenotypes (Chemotypes) in European Agricultural Hemp (Cannabis sativa L.) Cultivars

In Europe, more than 50 approved cultivars of fiber hemp (Cannabis sativa L.) are in agricultural production. Their content of psychoactive tetrahydrocannabinol (THC) is legally restricted to <0.2% (%w/w in the dry, mature inflorescences). Cannabis strains with much higher THC contents are also grown, illegally or under license for drug production. Differentiation between these two groups relies on biochemical quantification of cannabinoid contents in mature floral material. For nonflowering material or tissue devoid of cannabinoids, the genetic prediction of the chemical phenotype (chemotype) provides a suitable method of distinction. Three discrete chemotypes, depending on the ratio of THC and the noneuphoric cannabidiol (CBD), can be distinguished: a “THC-predominant” type, a “CBD-predominant” type, and an intermediate chemotype. We present a systematic genetic prediction of chemotypes of 62 agricultural hemp cultivars grown in Europe. The survey reveals the presence of up to 35% B_T allele-carrying individuals (representing either a THC-predominant or an intermediate chemotype) in some cultivars—which is unexpected considering the legal THC limit of 0.2% THC. The fact that 100% of the seized drug-type seeds in this study revealed at least one B_T allele, reflects that plant breeding efforts have resulted in a fixation of the B_T allele in recreational Cannabis. To guarantee a sincere forensic application based on a genetic chemotype prediction, we recommend not to classify material of unknown origin if the samples size is below nine genetically independent individuals.